Cell-cell signaling pathways, such as Wnt, Notch, and MAPK, are critical for the proper fate specification of most cells and tissues during animal development. These ancient and highly conserved pathways regulate developmental cell fate and adult health primarily by altering the activity of transcription factors (TFs), which in turn control the expression of target genes. Signal-regulated enhancers (or cis-regulatory elements) located near pathway target genes contain binding sites for signal-regulated TFs and for other, tissue- or cell-type- specific TFs;interactions among these factors determine the expression pattern of the target gene. However, no signal-regulated enhancer in any multicellular organism has been fully characterized, in the sense that all of its necessary direct regulatory inputs have been accounted for. Furthermore, at the mechanistic level, none of the known biochemical activities implicated in transcriptional activation have been shown to be sufficient, alone or together, to drive gene expression during animal development. These and other gaps in our understanding suggest that important mechanisms of transcriptional activation are likely still undiscovered. The proposed research program is dedicated to "solving" two signal-regulated developmental enhancers;that is, to defining and functionally characterizing all of their required regulatory sub-elements. Our results, along with other studies, show definitively that simply combining the known regulatory TF sites of well-characterized enhancers is not sufficient to stimulate transcription in vivo. The project proposed here combines in vivo functional reporter assays with the investigation of known transcriptional control mechanisms, the identification of novel regulators, and the advantages of Drosophila genetics. The project has two main objectives: (1) Identify the DNA elements within the sparkling (spa) and dppVM enhancers that are necessary for gene activation in vivo;determine the topological/structural rules for arrangement of those elements. (2) Determine the roles of the known and novel regulatory elements within spa and dppVM in mechanisms of transcriptional control such as local chromatin modification, cooperative DNA binding, nucleosome positioning, and intra-nuclear organization;isolate and characterize factors binding to novel regulatory DNA elements within these enhancers. Project Narrative The signaling pathways and factors to be studied are critical for normal animal development, and are implicated in a host of developmental defects and human diseases, most notably cancer. A better mechanistic understanding of signal-regulated enhancers will facilitate the development of strategies for the treatment and prevention of disorders caused by aberrant cell signaling and gene expression, and may further the development of new molecular tools with significant clinical and research utility.